Neutrophile et immunité

Le polynucléaire neutrophile, longtemps considéré comme une simple cellule phagocytaire à durée de vie très courte éliminant les intrus de l’organisme, est, en fait, une cellule dont les diverses facettes fonctionnelles connues en font un acteur indispensable des défenses de première ligne, certes, mais aussi des défenses acquises. À cet aspect multifonctionnel s’associent un retard de sa mort programmée, une plasticité éclairant sa capacité à se transdifférencier en fonction des besoins locaux du processus inflammatoire et infectieux, mais aussi une hétérogénéité de cette cellule constituant des sous-populations variables en fonction des individus. Le neutrophile est certainement la cellule hématopoïétique qui suscite le plus de controverses, dont bien des propriétés restent à découvrir, et dont les secrets fonctionnels cachent sans doute d’étonnantes capacities

SARS-CoV-2 Nsp2 Contributes to Inflammation by Activating NF-κB

COVID-19 is associated with robust inflammation and partially impaired antiviral responses. The modulation of inflammatory gene expression by SARS-CoV-2 is not completely understood. In this study, we characterized the inflammatory and antiviral responses mounted during SARS-CoV-2 infection. K18-hACE2 mice were infected with a Wuhan-like strain of SARS-CoV-2, and the transcriptional and translational expression interferons (IFNs), cytokines, and chemokines were analyzed in mouse lung homogenates. Our results show that the infection of mice with SARS-CoV-2 induces the expression of several pro-inflammatory CC and CXC chemokines activated through NF-κB but weakly IL1β and IL18 whose expression are more characteristic of inflammasome formation. We also observed the downregulation of several inflammasome effectors. The modulation of innate response, following expressions of non-structural protein 2 (Nsp2) and SARS-CoV-2 infection, was assessed by measuring IFNβ expression and NF-κB modulation in human pulmonary cells. A robust activation of the NF-κB p65 subunit was induced following the infection of human cells with the corresponding NF-κB-driven inflammatory signature. We identified that Nsp2 expression induced the activation of the IFNβ promoter through its NF-κB regulatory domain as well as activation of p65 subunit phosphorylation. The present studies suggest that SARS-CoV-2 skews the antiviral response in favor of an NF-κB-driven inflammatory response, a hallmark of acute COVID-19 and for which Nsp2 should be considered an important contributor

Effects of signaling inhibitors on IL-32γ delayed apoptosis.

<p>IL-32γ-delayed apoptosis is dependent on kinase activation. Neutrophils were pre-incubated 30 min with an inhibitor of PI-3 kinase (LY249002 at 20 µM), an inhibitor of MEK (U0126 at 10 µM), an inhibitor of p38 MAP kinase (SB202190 at 25 µM), and an inhibitor of JN Kinase (SP600125 at 10 µM) or their vehicle (DMSO). Neutrophils were then stimulated with IL-32γ (300 ng.ml⁻¹) for 20 h and further analyzed by FACS to evaluate their annexin V-FITC binding in conjunction with propidium iodide staining. The effects of signaling inhibitors were compared to the experimental condition in which neutrophils were incubated with IL-32γ + inhibitor vehicle (DMSO); this condition corresponds to 100% of delayed apoptosis. Results are expressed as percentage of IL-32γ inhibitory effect. Results are means ± s.e.m. of 5–9 different donors. Statistics: paired Student <i>t</i> test, p values are indicated in the table. NS  =  non-significant.</p><p>Effects of signaling inhibitors on IL-32γ delayed apoptosis.</p

IL-32γ delays neutrophil apoptosis.

<p>Neutrophils were incubated with graded concentrations of IL-32γ or its vehicle. Cells were analyzed by flow cytometry for annexin V-FITC binding in conjunction with propidium iodide (PI) staining. Representative FACS analysis of neutrophils incubated for 24 hrs with vehicle (Control) or 300 ng.ml⁻¹ of IL-32γ and stained with annexin V-FITC and PI (<b>A</b>). Neutrophils were incubated with vehicle (0) or graded concentrations of IL-32γ (10, 30, 100, 300 and 1000 ng.ml⁻¹) for 24 hrs (<b>B</b>) or 48 hrs (<b>C</b>). Results are illustrated in (A) as annexin V-FITC negative and PI negative cells (non-apoptotic cells; present in the lower left quadrant of FACS), annexin V-FITC positive and PI negative cells (early apoptotic cells; lower right quadrant), annexin V-FITC positive and PI positive cells (late apoptotic cells; upper right quadrant) or annexin V-FITC negative and PI positive cells (necrotic cells; upper left quadrant). Apoptosis was also evaluated by measurement of neutrophil pan-caspase activity after 48 hrs of incubation with IL-32γ or its vehicle. Activated caspases were labelled with a FITC-conjugated pan-caspase inhibitor before being analyzed by FACS (<b>D</b>). Results represent the means ± s.e.m. of 4 different donors. Statistical significance was assessed using one-way ANOVA followed by Bonferroni multiple comparison test: means without a common letter differ (P<0.05).</p

IL-32γ induces sustained steady state levels of MCL-1 protein.

<p>Human neutrophils were incubated for 8, 20 and 30 hrs with IL-32γ (300 ng.ml⁻¹) or its vehicle (absence of IL-32γ). Cell lysates were subjected to SDS-PAGE and transferred to PVDF membranes. Western blotting was then performed with anti-MCL-1 or anti-β-actin (control of protein loading) antibodies, and proteins were revealed with Western Lightning Plus ECL. Representative of 4 different donors (<b>A</b>). Densitometric analyses of MCL-1 and actin expression were performed and ratios between MCL-1 and actin were calculated to normalize the data. These normalized values were then used to calculate the percentages of MCL-1 variations in neutrophils incubated with vehicle at 20 and 30 hrs of incubation or with IL-32γ at 8, 20 and 30 hrs of incubation, considering that the normalized value of MCL-1 in neutrophils incubated with vehicle at 8 hrs represents 100% MCL-1 (neutrophils at 8 hrs of incubation with vehicle were 96% non-apoptotic) (<b>B</b>). Results are means ± s.e.m. of 4 different donors. Statistics: paired Student <i>t</i> test, * p<0.05.</p

IL-32γ induces phosphorylation of p38 MAPK in human blood neutrophils.

<p>Freshly isolated neutrophils were incubated in the presence of IL-32γ (500 ng.ml⁻¹) or its vehicle (HBSS) for the indicated time. Phosphorylation of p38 and total p38 were detected by immunoblot. Representative of 4 different donors.</p